Survey of drug use and its association with herd-level and farm-level characteristics on German dairy farms.

The use of veterinary drugs is of similar importance to that of human drugs in addressing health challenges. In this context, pharmaceuticals and their metabolites inevitably enter soil and water in unknown quantities. Therefore, this study collects and analyzes drug data from 2020 for 50 dairy farms located in Germany. The most frequently used substance group is antibiotics (40.13%), followed by antiphlogistics (18.86%), antiparasitics (13.09%), and hormones (9.29%). Treatment frequencies record the number of days per year on which an average animal on a farm was treated with a substance. The calculated values range from 0.94 to 21.69 d/yr and are distributed heterogeneously across farms. In this study, on average, a cow was treated on 6 d in 2020: 2.34 d with antibiotics, 1.07 d with antiphlogistics, 0.76 d with antiparasitics, and 0.41 d with hormones. In addition to individual farm management practices, other factors are related to treatment frequency. Farms with a veterinary care contract used more hormonal substances than farms without a care contract. In addition, higher milk yield coincides with more frequent treatments with antiphlogistic or hormonal substances. Other related factors include grazing, longevity, farm size, and use of a claw bath. Our study represents an important first step in describing the amounts and determinants of veterinary drugs used in livestock farming. Such insights on magnitudes and farm parameters are essential to estimate potential environmental effects and derive strategies to reduce veterinary drug use.

High temperatures augment inhibition of parasites by a honey bee gut symbiont.

Temperature affects growth, metabolism, and interspecific interactions in microbial communities. Within animal hosts, gut bacterial symbionts can provide resistance to parasitic infections. Both infection and populations of symbionts can be shaped by the host body temperature. However, the effects of temperature on the antiparasitic activities of gut symbionts have seldom been explored. The Lactobacillus-rich gut microbiota of facultatively endothermic honey bees is subject to seasonal and ontogenetic changes in host temperature that could alter the effects of symbionts against parasites. We used cell cultures of a Lactobacillus symbiont and an important trypanosomatid gut parasite of honey bees to test the potential for temperature to shape parasite-symbiont interactions. We found that symbionts showed greater heat tolerance than parasites and chemically inhibited parasite growth via production of acids. Acceleration of symbiont growth and acid production at high temperatures resulted in progressively stronger antiparasitic effects across a temperature range typical of bee colonies. Consequently, the presence of symbionts reduced both the peak growth rate and heat tolerance of parasites. Substantial changes in parasite-symbiont interactions were evident over a temperature breadth that parallels changes in diverse animals exhibiting infection-related fevers and the amplitude of circadian temperature variation typical of endothermic birds and mammals, implying the frequent potential for temperature to alter symbiont-mediated resistance to parasites in endo- and ectothermic hosts. Results suggest that the endothermic behavior of honey bees could enhance the impacts of gut symbionts on parasites, implicating thermoregulation as a reinforcer of core symbioses and possibly microbiome-mediated antiparasitic defense. IMPORTANCE Two factors that shape the resistance of animals to infection are body temperature and gut microbiota. However, temperature can also alter interactions among microbes, raising the question of whether and how temperature changes the antiparasitic effects of gut microbiota. Honey bees are agriculturally important hosts of diverse parasites and infection-mitigating gut microbes. They can also socially regulate their body temperatures to an extent unusual for an insect. We show that high temperatures found in honey bee colonies augment the ability of a gut bacterial symbiont to inhibit the growth of a common bee parasite, reducing the parasite's ability to grow at high temperatures. This suggests that fluctuations in colony and body temperatures across life stages and seasons could alter the protective value of bees' gut microbiota against parasites, and that temperature-driven changes in gut microbiota could be an underappreciated mechanism by which temperature-including endothermy and fever-alters animal infection.

Targeting Toxoplasma gondii ME49 TgAPN2: A Bioinformatics Approach for Antiparasitic Drug Discovery.

As fewer therapeutic options are available for treating toxoplasmosis, newer antiparasitic drugs that can block TgAPN2 M1 aminopeptidase are of significant value. Herein, we employed several computer-aided drug-design approaches with the objective of identifying drug molecules from the Asinex library with stable conformation and binding energy scores. By a structure-based virtual screening process, three molecules-LAS_52160953, LAS_51177972, and LAS_52506311-were identified as promising candidates, with binding affinity scores of -8.6 kcal/mol, -8.5 kcal/mol, and -8.3 kcal/mol, respectively. The compounds produced balanced interacting networks of hydrophilic and hydrophobic interactions, vital for holding the compounds at the docked cavity and stable binding conformation. The docked compound complexes with TgAPN2 were further subjected to molecular dynamic simulations that revealed mean RMSD for the LAS_52160953 complex of 1.45 Å), LAS_51177972 complex 1.02 Å, and LAS_52506311 complex 1.087 Å. Another round of binding free energy validation by MM-GBSA/MM-PBSA was done to confirm docking and simulation findings. The analysis predicted average MM-GBSA value of <-36 kcal/mol and <-35 kcal/mol by MM-PBSA. The compounds were further classified as appropriate candidates to be used as drug-like molecules and showed favorable pharmacokinetics. The shortlisted compounds showed promising biological potency against the TgAPN2 enzyme and may be used in experimental validation. They may also serve as parent structures to design novel derivatives with enhanced biological potency.

Microbe Profile: Wigglesworthia glossinidia: the tsetse fly's significant other.

Wigglesworthia glossinidia is an obligate, maternally transmitted endosymbiont of tsetse flies. The ancient association between these two organisms accounts for many of their unique physiological adaptations. Similar to other obligate mutualists, Wigglesworthia's genome is dramatically reduced in size, yet it has retained the capacity to produce many B-vitamins that are found at inadequate quantities in the fly's vertebrate blood-specific diet. These Wigglesworthia-derived B-vitamins play essential nutritional roles to maintain tsetse's physiological homeostasis as well as that of other members of the fly's microbiota. In addition to its nutritional role, Wigglesworthia contributes towards the development of tsetse's immune system during the larval period. Tsetse produce amidases that degrade symbiotic peptidoglycans and prevent activation of antimicrobial responses that can damage Wigglesworthia. These amidases in turn exhibit antiparasitic activity and decrease tsetse's ability to be colonized with parasitic trypanosomes, which reduce host fitness. Thus, the Wigglesworthia symbiosis represents a fine-tuned association in which both partners actively contribute towards achieving optimal fitness outcomes.

Bioactive Lipodepsipeptides Produced by Bacteria and Fungi.

Natural products are a vital source for agriculture, medicine, cosmetics and other fields. Lipodepsipeptides (LPDs) are a wide group of natural products distributed among living organisms such as bacteria, fungi, yeasts, virus, insects, plants and marine organisms. They are a group of compounds consisting of a lipid connected to a peptide, which are able to self-assemble into several different structures. They have shown different biological activities such as phytotoxic, antibiotic, antiviral, antiparasitic, antifungal, antibacterial, immunosuppressive, herbicidal, cytotoxic and hemolytic activities. Their biological activities seem to be due to their interactions with the plasma membrane (MP) because they are able to mimic the architecture of the native membranes interacting with their hydrophobic segment. LPDs also have surfactant properties. The review has been focused on the lipodepsipeptides isolated from fungal and bacterial sources, on their biological activity, on the structure-activity relationships of some selected LPD subgroups and on their potential application in agriculture and medicine. The chemical and biological characterization of lipodepsipeptides isolated in the last three decades and findings that resulted from SCI-FINDER research are reported. A critical evaluation of the most recent reviews dealing with the same argument has also been described.

The effects of teflubenzuron on mortality, physiology and accumulation in Capitella sp.

The chitin synthesis inhibitor teflubenzuron (TFB) is a feed antiparasitic agents used to impede molting of the salmon lice, an ecto-parasite that severely affects the salmon industry. Low absorption of oral administered TFB may cause elevated concentrations in the feces discharged from the salmon into the benthic environment. The polychaete Capitella sp. are often dominant in such habitats and consume organic waste deposited on the sediment. In the present study, Capitella sp. were exposed to doses of TFB in salmon feed of 1, 2 and 4 g TFB kg-1 (0 g TFB kg-1 in control group) over an experimental period of 32 days. Cumulative mortality was 12%-15% in both treatment groups with 1 and 2 g TFB kg-1 and reached 27% in the group with 4 g TFB kg-1. Only the highest dose (4 g TFB kg-1) negatively affected feed intake, growth and respiration of the polychaetes while food conversion efficiency was not affected. At the end of the experiment, the concentrations of TFB in the Capitella sp. were high, in the range of 9.24-10.32 µg g-1 for the three treatment groups. It was suggested that a maximum level of absorption rate was reached, also for the lowest dose. High concentrations of TFB in the Capitella sp. might pose a risk to crustaceans that forage for polychaetes in the vicinity of fish farms. We conclude that the effects of TFB on Capitella sp. may therefore primarily be to the predators rather than the Capitella sp.

Functional Characterization of Temporin-SHe, a New Broad-Spectrum Antibacterial and Leishmanicidal Temporin-SH Paralog from the Sahara Frog (Pelophylax saharicus).

Amphibian skin is a promising natural resource for antimicrobial peptides (AMPs), key effectors of innate immunity with attractive therapeutic potential to fight antibiotic-resistant pathogens. Our previous studies showed that the skin of the Sahara Frog (Pelophylax saharicus) contains broad-spectrum AMPs of the temporin family, named temporins-SH. Here, we focused our study on temporin-SHe, a temporin-SHd paralog that we have previously identified in this frog but was never structurally and functionally characterized. We synthesized and determined the structure of temporin-SHe. This non-amphipathic α-helical peptide was demonstrated to strongly destabilize the lipid chain packing of anionic multilamellar vesicles mimicking bacterial membranes. Investigation of the antimicrobial activity revealed that temporin-SHe targets Gram-negative and Gram-positive bacteria, including clinical isolates of multi-resistant Staphylococcus aureus strains. Temporin-SHe exhibited also antiparasitic activity toward different Leishmania species responsible for visceral leishmaniasis, as well as cutaneous and mucocutaneous forms. Functional assays revealed that temporin-SHe exerts bactericidal effects with membrane depolarization and permeabilization, via a membranolytic mechanism observed by scanning electron microscopy. Temporin-SHe represents a new member of the very limited group of antiparasitic temporins/AMPs. Despite its cytotoxicity, it is nevertheless an interesting tool to study the AMP antiparasitic mechanism and design new antibacterial/antiparasitic agents.

Secondary Metabolites and Their Bioactivities Produced by Paecilomyces.

Paecilomyces, a common saprobic filamentous fungus, not only plays an important role in biological control, but also has applications in medicine, food, and environmental protection. In this paper, 223 secondary metabolites and their bioactivities from 13 known species and various unidentified strains of Paecilomyces are reviewed. Their structures can be described as polyketide, terpenoid, peptide, alkaloid, quinone, pyrone, sterol, and fatty acid. They have been demonstrated varying biological activities, including antimicrobial, antitumor, insecticidal, antiplasmodial, antimalarial, nematicidal, herbicidal, and enzyme-inhibiting. This review provides a comprehensive overview of secondary metabolites and their biological activities from strains of Paecilomyces.

A Single Amino Acid Substitution in the Third Transmembrane Region Has Opposite Impacts on the Selectivity of the Parasiticides Fluralaner and Ivermectin for Ligand-Gated Chloride Channels.

Fluralaner (Bravecto) is a recently marketed isoxazoline ectoparasiticide. This compound potently inhibits GABA-gated chloride channels (GABACls) and less potently glutamate-gated chloride channels (GluCls) in insects. The mechanism underlying this selectivity is unknown. Therefore, we sought to identify the amino acid residues causing the low potency of fluralaner toward GluCls. We examined the fluralaner sensitivity of mutant housefly (Musca domestica) GluCls in which amino acid residues in the transmembrane subunit interface were replaced with the positionally equivalent amino acids of Musca GABACls. Of these amino acids, substitution of an amino acid (Leu315) in the third transmembrane region (TM3) with an aromatic amino acid dramatically enhanced the potency of fluralaner in the GluCls. In stark contrast to the enhancement of fluralaner potency, this mutation eliminated the activation of currents and the potentiation but not the antagonism of glutamate responses that are otherwise all elicited by the macrolide parasiticide ivermectin (IVM). Our findings indicate that the amino acid Leu315 in Musca GluCls plays significant roles in determining the selectivity of fluralaner and IVM for these channels. Given the high sequence similarity of TM3, this may hold true more widely for the GluCls and GABACls of other insect species.

Asymmetric synthesis and evaluation of epoxy-α-acyloxycarboxamides as selective inhibitors of cathepsin L.

Cathepsin L plays important roles in physiological processes as well as in the development of many pathologies. Recently the attentions were turned to its association with tumor progress what makes essential the development of more potent and selective inhibitors. In this work, epoxipeptidomimetics were investigated as new cathepsin inhibitors. This class of compounds is straightforward obtained by using a green one-pot asymmetric epoxidation/Passerini 3-MCR. A small library of 17 compounds was evaluated against cathepsin L, and among them LSPN423 showed to be the most potent. Investigations of the mechanism suggested a tight binding uncompetitive inhibition.

Structure and Biosynthesis of Isatropolones, Bioactive Amine-Scavenging Fluorescent Natural Products from Streptomyces†Gö66.

The natural products isatropolone A-C (1-3) were reisolated from Streptomyces Gö66, with 1 and 3 showing potent activity against Leishmania donovani. They contain a rare tropolone ring derived from a type II polyketide biosynthesis pathway. Their biosynthesis was elucidated by labeling experiments, analysis of the biosynthesis gene cluster, its partial heterologous expression, and structural characterization of various intermediates. Owing to their 1,5-diketone moiety, they can react with ammonia, amines, lysine, and lysine-containing peptides and proteins, which results in the formation of a covalent bond and subsequent pyridine ring formation. Their fluorescence properties change upon amine binding, enabling the simple visualization of reacted amines including proteins.

N-myristoyltransferase inhibitors as new leads to treat sleeping sickness.

African sleeping sickness or human African trypanosomiasis, caused by Trypanosoma brucei spp., is responsible for approximately 30,000 deaths each year. Available treatments for this disease are poor, with unacceptable efficacy and safety profiles, particularly in the late stage of the disease when the parasite has infected the central nervous system. Here we report the validation of a molecular target and the discovery of associated lead compounds with the potential to address this lack of suitable treatments. Inhibition of this target-T. brucei N-myristoyltransferase-leads to rapid killing of trypanosomes both in vitro and in vivo and cures trypanosomiasis in mice. These high-affinity inhibitors bind into the peptide substrate pocket of the enzyme and inhibit protein N-myristoylation in trypanosomes. The compounds identified have promising pharmaceutical properties and represent an opportunity to develop oral drugs to treat this devastating disease. Our studies validate T. brucei N-myristoyltransferase as a promising therapeutic target for human African trypanosomiasis.

In Vitro Effect of the Synthetic cal14.1a Conotoxin, Derived from Conus californicus, on the Human Parasite Toxoplasma gondii.

Toxins that are secreted by cone snails are small peptides that are used to treat several diseases. However, their effects on parasites with human and veterinary significance are unknown. Toxoplasma gondii is an opportunistic parasite that affects approximately 30% of the world's population and can be lethal in immunologically compromised individuals. The conventional treatment for this parasitic infection has remained the same since the 1950s, and its efficacy is limited to the acute phase of infection. These findings have necessitated the search for new drugs that specifically target T. gondii. We examined the effects of the synthetic toxin cal14.1a (s-cal14.1a) from C. californicus on the tachyzoite form of T. gondii. Our results indicate that, at micromolar concentrations, s-cal14.1a lowers viability and inhibits host cell invasion (by 50% and 61%, respectively) on exposure to extracellular parasites. Further, intracellular replication decreased significantly while viability of the host cell was unaffected. Our study is the first report on the antiparasitic activity of a synthetic toxin of C. californicus.

Simultaneous quantification of tizoxanide and tizoxanide glucuronide in mouse plasma by liquid chromatography-tandem mass spectrometry.

Nitazoxanide (NTZ) is a broad-spectrum antimicrobial agent. Tizoxanide (T) and tizoxanide glucuronide (TG) are the major circulating metabolites after oral administration of NTZ. A rapid and specific LC-MS/MS method for the simultaneous quantification of T and TG in mouse plasma was developed and validated. A simple acetonitrile-induced protein precipitation method was employed to extract two analytes and the internal standard glipizide from 50 µL of mouse plasma. The purified samples were resolved using a C18 column with a mobile phase consisting of acetonitrile and 5 mm ammonium formate buffer (containing 0.05% formic acid) following a gradient elution. An API 3000 triple quadrupole mass spectrometer was operated under multiple reaction-monitoring mode with electrospray ionization. The precursor-to-product ion transitions m/z 264 → m/z 217 for T and m/z 440 → m/z 264 for TG were used for quantification. The developed method was linear in the concentration ranges of 1.0-500.0 ng/mL for T and 5.0-1000.0 ng/mL for TG. The intra- and inter-day precision and accuracy of the quality control samples at low, medium and high concentrations exhibited an RSD of <13.2% and the accuracy values ranged from -9.6 to 9.3%. We used this validated method to study the pharmacokinetics of T and TG in mice following oral administration of NTZ. Copyright © 2016 John Wiley & Sons, Ltd.

Marine Pyridoacridine Alkaloids: Biosynthesis and Biological Activities.

Pyridoacridines are a class of strictly marine-derived alkaloids that constitute one of the largest chemical families of marine alkaloids. During the last few years, both natural pyridoacridines and their analogues have constituted excellent targets for synthetic works. They have been the subject of intense study due to their significant biological activities; cytotoxic, antibacterial, antifungal, antiviral, insecticidal, anti-HIV, and anti-parasitic activities. In the present review, 95 pyridoacridine alkaloids isolated from marine organisms are discussed in term of their occurrence, biosynthesis, biological activities, and structural assignment.

Comparative plasma and milk dispositions, faecal excretion and efficacy of per os ivermectin and pour-on eprinomectin in horses.

The horse milk gains increasing interest as a food product for sensitive consumers, such as children with food allergies or elderly people. We investigated the plasma and milk disposition, faecal excretion and efficacy of per os ivermectin (IVM) and pour-on eprinomectin (EPM) in horses. Ten mares were divided into two groups. The equine paste formulation of IVM and bovine pour-on formulation of EPM were administered orally and topically at dosage of 0.2 and 0.5 mg/kg bodyweight. Blood, milk and faecal samples were analysed using high-performance liquid chromatography. The plasma concentration and persistence of IVM were significantly greater and longer compared with those of EPM. Surprisingly, EPM displayed a much higher disposition rate into milk (AUCmilk/plasma : 0.48) than IVM (AUCmilk/plasma : 0.19). IVM exhibited significantly higher faecal excretion (AUCfaeces : 7148.54 ng·d/g) but shorter faecal persistence (MRTfaeces : 1.17 days) compared with EPM (AUCfaeces : 42.43 ng·d/g and MRTfaeces : 3.29 days). Faecal strongyle egg counts (EPG) were performed before and at weekly intervals after treatment. IVM reduced the EPG by 96-100% for up to 8 weeks, whereas the reduction in the EPM group varied from 78 to 99%. In conclusion, due to the relatively low excretion in milk, EPM and IVM may be used safely in lactating mares if their milk is used for human consumption. Nevertheless, much lower plasma and faecal availabilities of EPM could result in subtherapeutic concentrations, which may increase the risk of drug resistance in nematodes after pour-on EPM administration compared with per os IVM.

Brain penetration of emodepside is increased in P-glycoprotein-deficient mice and leads to neurotoxicosis.

The antiparasitic drug emodepside (EMO) is a substrate of the P-glycoprotein multidrug efflux carrier (P-gp; syn. MDR1, ABCB1), which has an important function in protecting the brain from potentially toxic compounds by functional drug efflux at the blood-brain barrier (BBB). Many dogs of the Collie breed and even dogs of many other breeds have a loss-of-function 4-bp deletion mutation in the MDR1 gene. In these dogs, brain penetration of many P-gp-transported drugs is increased and so their therapeutic usage is restricted. To elucidate the role of P-gp at the BBB for the brain penetration of EMO, we applied EMO at 1 mg/kg to mdr1-deficient (PGP(mut) ) and mdr1-intact (PGP(WT) ) CF1 mice. Whereas in the brain of the PGP(WT) mice, EMO was below the detection level of 10 ng/g, its concentration was at 43.7 ng/g in the PGP(mut) mice. Furthermore, appearance of neurological toxicity was analyzed in these mice after application of 1 mg/kg EMO using a rotarod setup. In all PGP(mut) mice, but not in the PGP(WT) mice, the walking performance on the rotarod was impaired by EMO with clear differences in the degree and duration of neurological toxicity. Some of the mice were completely unable to walk on the rotarod already at 2 h after drug application and showed long-lasting ataxia over >24 h. Others even showed significantly reduced walking performance, but completely recovered within 1 day. In conclusion, P-gp restricts brain penetration of EMO and prevents neurological toxicity of this drug in mice.

Engineering of the TetR family transcriptional regulator SAV151 and its target genes increases avermectin production in Streptomyces avermitilis.

Avermectins produced by Streptomyces avermitilis are used commercially for broad-spectrum parasite control in medical, veterinary, and agricultural fields. Our previous comparative transcriptome analysis of wild-type strain ATCC31267 vs. avermectin-overproducing strain 76-02-e revealed that the gene SAV151, which encodes a TetR family transcriptional regulator, was downregulated in 76-02-e. In the present study, we investigated the role of SAV151 in avermectin production. Deletion of SAV151 increased avermectin yield ~1-fold in ATCC31267, and this phenotype was complemented by a single copy of SAV151. Overexpression of SAV151 in ATCC31267 reduced avermectin yield by ~70%. RT-PCR analysis showed that the promoting effect of SAV151 deletion on avermectin production was not due to alteration of ave genes at the transcriptional level. SAV151 negatively regulated the transcription of itself and of the adjacent transcriptional unit SAV152-SAV153-SAV154. In chromatin immunoprecipitation and gel shift assays, purified His6-tagged SAV151 protein bound to the bidirectional SAV151-SAV152 promoter region. SAV151 bound to two palindromic sequences in this region and thereby repressed transcription from both directions. Two of the SAV151 target genes, SAV152 (which encodes a putative dehydrogenase) and SAV154 (which encodes a putative hydrolase), had promoting effects on avermectin production. Our findings provide the basis for a strategy to increase avermectin production by controlling SAV151 and its target genes.

Activation of complement-like antiparasitic responses in Anopheles mosquitoes.

During their development in mosquitoes, malaria parasites undergo massive losses that are in part due to a potent antiparasitic response mounted by the vector. The most efficient and best-characterized response relies on a complement-like system particularly effective against parasites as they cross the mosquito midgut epithelium. While our vision of the molecular and cellular events that lead to parasite elimination is still partial, our understanding of the steps triggering complement activation at the surface of invading parasites has considerably progressed, not only through the identification of novel contributing genes, but also with the recent in-depth characterization of the different mosquito blood cell types, and the ability to track them in live mosquitoes. Here, we propose a simple model based on the time of invasion to explain how parasites may escape complement-like responses during midgut infection.

Cryptosporidium uses CSpV1 to activate host type I interferon and attenuate antiparasitic defenses.

Cryptosporidium infects gastrointestinal epithelium and is a leading cause of infectious diarrhea and diarrheal-related death in children worldwide. There are no vaccines and no fully effective therapy available for the infection. Type II and III interferon (IFN) responses are important determinants of susceptibility to infection but the role for type I IFN response remains obscure. Cryptosporidium parvum virus 1 (CSpV1) is a double-stranded RNA (dsRNA) virus harbored by Cryptosporidium spp. Here we show that intestinal epithelial conditional Ifnar1-/- mice (deficient in type I IFN receptor) are resistant to C. parvum infection. CSpV1-dsRNAs are delivered into host cells and trigger type I IFN response in infected cells. Whereas C. parvum infection attenuates epithelial response to IFN-γ, loss of type I IFN signaling or inhibition of CSpV1-dsRNA delivery can restore IFN-γ-mediated protective response. Our findings demonstrate that type I IFN signaling in intestinal epithelial cells is detrimental to intestinal anti-C. parvum defense and Cryptosporidium uses CSpV1 to activate type I IFN signaling to evade epithelial antiparasitic response.